We tested for HI antibodies to akabane virus and arbor virus in the serums of mothers of affected children (all babies died) born at Bega, and a group confined of children with similar abnormalities in Sydney, and a control group for both areas who had had normal children at the same time. Human serums proved cytotoxic for the cells using the microtitre neutralisation test and the test was carried out in roller tubes, as described by Snowdon ( 1 9 7 0 ) . Verotube cultures for antibody t o akabane virus were negative for all serums tested. A low titre ( 4 0 ) for Ross River alphavirus was detected in the serum of a mother of an affected child in Sydney and one control mother from Sydney gave a low titre reactivity ( 2 0 ) for flavivirus (Murray Valley encephalitis virus). There was, therefore, no evidence of involvement of these viruses by maternal infection, as the teratogenic agent in neural tube closure defects.
I wish to thank Mr R. A. Wanner, Department of Veterinary Medicine, Camden, and Mr A. J. Della Porta and Dr W. A. Snowdon at the CSIRO Division of Animal Health, Parkville, Victoria, and Dr A. Brown, Bega, for their help in this study. BARBARA FIELD, M.B., B.S. School of Public Health and Tropical Medicine, The University of Sydney, Sydney, New South Wales, 2006 14 November 1975 References Hartley, W. J. and Wanner, R. A. (1975)-Aust. vet. J . 51: 103. Leipold, H. W., Cates, W. F., Radostits, 0. M. and Howell, W. E. (1970)-Am. J . vet. Res. 31: 1367. Snowdon, W. A. (1970)-Aust. vet. J . 46: 358. Yen, S. and MacMahon, B. (1968)-Lancet ii: 623.
A RAPID ENZYMATIC TEST FOR THE ESTIMATION OF THE SOMATIC CELL COUNT IN BOVINE. MXLK
For a number of years now, researchers have been looking unsuccessfully for a rapid enzymatic test that would detect sub-clinical mastitis in bovine mammary glands (Cullen 1966; Patterson et a1 1969; Bogin and Ziv 1973). However, only one enzymatic procedure (catalase determination) has been used routinely to monitor abnormal udder secretions (Paape et a1 1965). This test is lengthy (2-3 h ) and it does not have a high correlation coefficient ( r = 0 . 4 9 ) with cell count (Janzen 1969). We wish to report in this letter an outline of a new enzymatic method for the estimation of somatic cells in bovine milk, and its advantages over currently used mastitis screening tests. The enzyme activity that was measured was Nacetyl-B-D-glucosaminidase (NAGase) . This enzyme appears to originate from cells and the technique indicated that the level of the enzyme is related to the cell count of the milk. The assay procedure was so designed that cell counts in the range 0-2 X 1O0/ml could be monitored. The assay procedure is as follows: 0.2 ml milk is mixed with 0.3 ml substrate solution ( 3 . 3 mM p-nitrophenyl-N-acetyl-B-D-glucosaminide in 0.33M citrate buffer pH 4.5) in a 15 mm X 15 cm test tube. The mixture is incubated for 15 min at 50°C. The reaction is stopped with 5.5 ml of 0 1M carbonate buffer pH 10. The released p-nitro phenol is measured with a h v i b o n d comparator using the same disc that is used in the alkaline phosphatase-pasteurisationtest. Interpretation of the t e s t Readings of 0, 6 or 10 indicate a cell count of < 0.5 X 1O0/ml while 10, 14, 18 or 25 indicate a cell count of > 0.5 X 10B/ml < 1.25 X 10'/ml. Some comments concerning this new test are as follows: It is rapid - 60-80 samples/hr for one operator. The overall test is inexpensive-it costs about $20 for substrate and buffers for 1000 tests. It is reliable - the correlation coefficient between comparator reading and electronic cell count was 0.87; on the same set of samples the correlation ooefficient between the Wisconsin Mastitis Test (WMT) and electronic cell count was 0.72. Also fewer false positive and false negative results were obtained than with the WMT procedure. 102
The procedure is simple and therefore a minimum of training is necessary. Milk samples can be analysed regardless of age and bacteriological quality. For example, milks can be stored at 5°C or at room temperature, with or without 0.1% formalin, for up to 10 days without significant effect on the comparator reading obtained. The ability of this test to use aged milk samples illustrates one of the important advantages it has over the WMT procedure (Postle 1971). Although it would be presumptuous to suggest that the NAGase procedure might at some stage displace the well tested and useful Wisconsin Mastitis Test, it should be. stressed that the enzymatic method is at least as good as and perhaps better than the WMT in a number of aspects. Nevertheless, the WMT procedure is faster and not as costly as the enzymatic method. However, this new test may well serve a useful purpose in the analysis of samples which have to be transported considerable distances to a centralised testing laboratory. Also, it may prove useful in testing bulk milks which are collected at greater than one day intervals, or it may be used in conjunction with other currently used screening tests in the role of a confirmatory diagnostic test. B. J. KITCHEN,M.Sc. G. MIDDLETON Otto Madsen Dairy Research Laboratory, Department of Primary Industries, 19 Hercules Street. Hamilton, Queensland, 4007 Id October 1975
References Bogin, E. and Ziv, G. (1973)-Corne11 V e t . 63: 666. Cullen, G. A. (1966)--Vet. Record 78: 277. Janzen, J. J. (1969)-J. Doiry Sci. 52: 329. Paape, M. I., Tucker, H. A. and Hafs, H. D. ( 1 9 6 5 ) J . Dairy Sci., 48: 191. Patterson, D. S. P., Berrett, S. and Cullen, G . A. ( 1 9 6 9 ) -Vet. Record 85: 708. Postle, D. S. (1971)-Ann. Meet. Nut. Mastitis Council ( U S A ) , p 25. Australian Veterinary Journal, Vol. 5 2 , February, 1976
I wish to thank Mr R. A. Wanner, Department of Veterinary Medicine, Camden, and Mr A. J. Della Porta and Dr W. A. Snowdon at the CSIRO Division of Animal Health, Parkville, Victoria, and Dr A. Brown, Bega, for their help in this study. BARBARA FIELD, M.B., B.S. School of Public Health and Tropical Medicine, The University of Sydney, Sydney, New South Wales, 2006 14 November 1975 References Hartley, W. J. and Wanner, R. A. (1975)-Aust. vet. J . 51: 103. Leipold, H. W., Cates, W. F., Radostits, 0. M. and Howell, W. E. (1970)-Am. J . vet. Res. 31: 1367. Snowdon, W. A. (1970)-Aust. vet. J . 46: 358. Yen, S. and MacMahon, B. (1968)-Lancet ii: 623.
A RAPID ENZYMATIC TEST FOR THE ESTIMATION OF THE SOMATIC CELL COUNT IN BOVINE. MXLK
For a number of years now, researchers have been looking unsuccessfully for a rapid enzymatic test that would detect sub-clinical mastitis in bovine mammary glands (Cullen 1966; Patterson et a1 1969; Bogin and Ziv 1973). However, only one enzymatic procedure (catalase determination) has been used routinely to monitor abnormal udder secretions (Paape et a1 1965). This test is lengthy (2-3 h ) and it does not have a high correlation coefficient ( r = 0 . 4 9 ) with cell count (Janzen 1969). We wish to report in this letter an outline of a new enzymatic method for the estimation of somatic cells in bovine milk, and its advantages over currently used mastitis screening tests. The enzyme activity that was measured was Nacetyl-B-D-glucosaminidase (NAGase) . This enzyme appears to originate from cells and the technique indicated that the level of the enzyme is related to the cell count of the milk. The assay procedure was so designed that cell counts in the range 0-2 X 1O0/ml could be monitored. The assay procedure is as follows: 0.2 ml milk is mixed with 0.3 ml substrate solution ( 3 . 3 mM p-nitrophenyl-N-acetyl-B-D-glucosaminide in 0.33M citrate buffer pH 4.5) in a 15 mm X 15 cm test tube. The mixture is incubated for 15 min at 50°C. The reaction is stopped with 5.5 ml of 0 1M carbonate buffer pH 10. The released p-nitro phenol is measured with a h v i b o n d comparator using the same disc that is used in the alkaline phosphatase-pasteurisationtest. Interpretation of the t e s t Readings of 0, 6 or 10 indicate a cell count of < 0.5 X 1O0/ml while 10, 14, 18 or 25 indicate a cell count of > 0.5 X 10B/ml < 1.25 X 10'/ml. Some comments concerning this new test are as follows: It is rapid - 60-80 samples/hr for one operator. The overall test is inexpensive-it costs about $20 for substrate and buffers for 1000 tests. It is reliable - the correlation coefficient between comparator reading and electronic cell count was 0.87; on the same set of samples the correlation ooefficient between the Wisconsin Mastitis Test (WMT) and electronic cell count was 0.72. Also fewer false positive and false negative results were obtained than with the WMT procedure. 102
The procedure is simple and therefore a minimum of training is necessary. Milk samples can be analysed regardless of age and bacteriological quality. For example, milks can be stored at 5°C or at room temperature, with or without 0.1% formalin, for up to 10 days without significant effect on the comparator reading obtained. The ability of this test to use aged milk samples illustrates one of the important advantages it has over the WMT procedure (Postle 1971). Although it would be presumptuous to suggest that the NAGase procedure might at some stage displace the well tested and useful Wisconsin Mastitis Test, it should be. stressed that the enzymatic method is at least as good as and perhaps better than the WMT in a number of aspects. Nevertheless, the WMT procedure is faster and not as costly as the enzymatic method. However, this new test may well serve a useful purpose in the analysis of samples which have to be transported considerable distances to a centralised testing laboratory. Also, it may prove useful in testing bulk milks which are collected at greater than one day intervals, or it may be used in conjunction with other currently used screening tests in the role of a confirmatory diagnostic test. B. J. KITCHEN,M.Sc. G. MIDDLETON Otto Madsen Dairy Research Laboratory, Department of Primary Industries, 19 Hercules Street. Hamilton, Queensland, 4007 Id October 1975
References Bogin, E. and Ziv, G. (1973)-Corne11 V e t . 63: 666. Cullen, G. A. (1966)--Vet. Record 78: 277. Janzen, J. J. (1969)-J. Doiry Sci. 52: 329. Paape, M. I., Tucker, H. A. and Hafs, H. D. ( 1 9 6 5 ) J . Dairy Sci., 48: 191. Patterson, D. S. P., Berrett, S. and Cullen, G . A. ( 1 9 6 9 ) -Vet. Record 85: 708. Postle, D. S. (1971)-Ann. Meet. Nut. Mastitis Council ( U S A ) , p 25. Australian Veterinary Journal, Vol. 5 2 , February, 1976
